Homeostasis of the organism depends upon interactions between protein-interacting modules and ligands to activate and deactivate cell signaling pathways for biological processes such as cell proliferation, cell death and protein degradation. Protein-interacting modules are conserved regions of amino acids that bind specific sequences in target proteins or position enzymes in close proximity to their substrates. For example, src homology domain 2 (SH2) binds phosphotyrosine residues on target cells to mediate receptor activation and receptor-ligand binding (Pawson, T., et al., Science 278:2075 (1997)). An example are WW-domains which are highly conserved regions of approximately 40 amino acids residues with two invariant tryptophans (W) in a triple stranded xcex2 sheet (Sudol, M. Prog. Biophys. Mol. Biol. 65:113 (1996); Rotin, D. Curr. Topics Microbiol. Immunol. 228:115 (1998)). Although the WW-domains of certain polypeptides have been implicated in protein-protein interactions by binding to proline rich sequences, many of their ligands do not contain proline rich sequences. (Sudol, M. Prog. Biophys. Mol. Biol. 65:113 (1996); Staub, O. et al., Structure 4:495 (1996), Rotin, D., Curr. Top. Microbiol. Immunol. 228:115 (1998)). Therefore, the role of WW-domain-containing proteins in mediating cell signaling events in biological processes is not known. However, due to their potential importance in cellular processes, it is important to elucidate a clearer understanding of the role of WW-domains in protein-protein interactions and cell signaling.
The present invention is based upon the discovery that WW-domains are phosphoserine or phosphothreonine binding modules. As further described herein, the present invention is also based upon the discovery that the WW-domain itself is phosphorylated, and that phosphorylation/dephosphorylation of the WW-domain polypeptide regulates the interaction of the WW-domain polypeptide with its phosphorylated ligand. As a result of this discovery, methods and compositions are available to modulate protein-protein interactions, e.g., the interaction between a signaling or regulatory polypeptide and its phosphorylated ligands.
The invention relates to methods of modulating protein-protein interactions comprising modulating the binding of WW-domain polypeptides with phosphorylated ligands. In one embodiment the binding interaction between the WW-domain containing polypeptide and phosphorylated ligand is inhibited. In another embodiment the binding interaction of the WW-domain containing polypeptide and phosphorylated ligand is enhanced. As used herein, a phosphorylated ligand is a molecule (e.g., protein, peptide, peptide mimetic or small organic molecule) containing a phosphoserine or phosphothreonine that binds to a WW-domain containing polypeptide. For example, ligands specifically encompassed by the present invention include tau protein, amyloid precursor protein, Cdc25C, Cdc27, Plk1, NIMA, Myt1, Rab4, Wee1, Mos, Sox3, Xbr1b, MP75 (E-MAP-115), MP110 (Cdc5), MP68, and MP30. WW-domain containing polypeptides specifically encompassed by the present invention include Pin1, NEDD4, YAP, FE65, formin binding protein, dystrophin, utropin, Ess1p/Ptf1p, Rsp5, Pub1, Dodo, Msb1, ORF1, YKB2, DP71, C38D4.5, P9659.21, Yo61, Yfx1, ZK1248.15, KO15c11, CD45AP, FBP11, FBP21, FBP23, FBP28 and FBP30.
Also encompassed by the present invention are molecules which mimic a WW-domain, referred to herein as WW-domain mimic molecules or pseudo-WW-domain molecules. Such molecules possess structural similarity with the WW-domains described herein or contain the consensus sequence LxxGWtx6Gtx(Y/F)(Y/F)h(N/D)Hx(T/S)tT(T/S)tWxtPt SEQ ID NO: 40 (where x=any amino acid, t=turn like or polar residue, and h=hydrophobic amino acid as described by Rotin, D., Curr. Top. Microbiol. Immunol. 228:115-133 (1998) the teachings of which are incorporated herein by reference in their entirety). For example, a WW-domain can contained the consensus sequence LPxGWExxxxxxxGxxYYxNHxTxxTxWxxP SEQ ID NO: 41, where x=any amino acid. The WW-domain mimic molecules are amino acid sequences, peptides, peptide mimetics, or polypeptides. The WW-domain mimic molecules are capable of interacting with, or binding to, phosphoserine/phosphothreonine ligands, thus modulating the activity of the phosphorylated ligand.
Also encompassed by the present invention are phosphorylated ligand sequences, referred to herein as phosphorylated ligand mimics, or phosphorylated pseudo-ligands. Phosphorylated ligand mimics are amino acid sequences, peptides, peptide mimetics, or polypeptides that contain a phosphoserine or phosphothreonine residue(s) and are of sufficient length and share sufficient amino acid identity with the ligand that the ligand mimics and interacts with, or binds to, the WW-domain containing polypeptide and thus modulates the activity of the WW-domain containing polypeptide.
A method of modulating the activity of a phosphorylated ligand or ligand mimic for a WW-domain, or a WW-domain containing polypeptide, comprises providing a WW-domain or WW-domain mimic which interacts with the ligand, wherein the activity of the phosphorylated ligand, ligand mimic, WW-domain polypeptide or WW-domain mimic is modulated (e.g., inhibited or enhanced). The activity can be binding activity between the ligand and WW-domain; enzymatic/regulatory activity of the WW-domain polypeptide; or both. For example, the prolyl-peptidyl cis-trans isomerase activity of Pin1 or ubiquitin ligase activity of Nedd4 can increase following binding of the WW-domain to a phosphorylated ligand.
Another aspect of the invention relates to regulating cell growth comprising mediating the binding of the WW-domain of Pin1 to a mitotic regulatory protein. The WW-domain can bind to a phosphorylated ligand (e.g., NIMA) resulting in cell proliferation. Cell proliferation can be regulated by regulating the phosphorylation state of the WW-domain. Dephosphorylation of the WW-domain of Pin1 leads to binding of the WW-domain to a phosphorylated ligand resulting in cell proliferation. Likewise, phosphorylation of the WW-domain inhibits binding to phosphorylated ligands resulting in cell death.
The invention also encompasses methods of regulating neurodegenerative diseases by modulating the interaction of a WW-domain and a ligand in cells (e.g., neurons, glial cells, Schwann cells) of the central (e.g., brain and spinal cord) and peripheral nervous system and any cells associated with the central or peripheral nervous systems (e.g., skeletal muscle). The interaction between the WW-domain and a neural cellular target can inhibit, halt, prevent or reverse neural degeneration by, for example, interfering with neuronal cell death (e.g., apoptosis, necrosis) or restoring neuronal function.
A further aspect of the invention encompasses a method of regulating the function of phosphorylated ligands of WW-domain containing polypeptides comprising mediating the binding of the ligand to the WW-domain. Specifically encompassed by the invention is a method of regulating the activity of hyperphosphorylated tau protein in Alzheimer""s disease comprising enhancing the binding of the WW-domain of Pin1 to the phosphorylated threonine 231 of tau whereby the binding of the WW-domain to tau results in binding of tau to microtubules leading to microtubule assembly. Another method of the invention relates to a method of regulating the interaction between the WW-domain of dystrophin and phosphorylated ligands.
The present invention further relates to a method of identifying a substance that modulates the interaction of a WW-domain containing polypeptide and a ligand, wherein the ligand is a phosphoserine or phosphothreonine ligand comprising contacting the WW-domain containing polypeptide with one, or more, test substances; maintaining the test substances and the WW-domain containing polypeptide under conditions suitable for interaction; and determining the interaction between the test substance and WW-domain containing polypeptide, wherein the interaction indicates that the test substance modulates the interaction between the WW-domain-containing polypeptide and the ligand. In one embodiment the interaction between the WW-domain and ligand that is modulated by the test substance is binding interaction. In another embodiment the interaction is enzymatic activity, in particular prolyl-peptidyl cis-trans isomerase activity of Pin1 or the ubiquitin ligase activity of Nedd4. The binding interaction or enzymatic activity between the WW-domain and ligand can be increased or decreased in the presence of the test substance. Thus, the test substance can be an antagonist or agonist of the interaction between the WW-domain and the ligand.
The present invention also provides mutants of WW-domain containing polypeptides comprising at least one mutation in the WW-domain. The ability of the mutant WW-domain containing polypeptides to bind a ligand is altered. In one embodiment the binding ability is enhanced. In another embodiment the binding ability is reduced. The mutant WW-domain containing polypeptides can also have altered enzymatic, catalytic or regulatory activity. In one embodiment the enzymatic activity of the WW-domain containing polypeptide is enhanced. In another embodiment the enzymatic activity is reduced. The mutant can have a mutation comprising a modification of an amino acid wherein the amino acid is selected from the group consisting of tyrosine at position 23, tryptophan at position 34, arginine at position 14, serine at position 16, serine at position 18 in Pin1, or equivalent positions in other WW-domain-containing proteins. The modified amino acid is replaced with an amino acid residue selected from the group consisting of alanine, glutamic acid or phenylalanine.
The invention also relates to a method of regulating protein degradation comprising regulating the phosphorylation of a serine residue of a WW-domain polypeptide. In particular, the WW-domain containing polypeptide is the ubiquitin ligase Nedd4. In one embodiment phosphorylation of the serine residue leads to binding of the WW-domain containing polypeptide and ligand to initiate polypeptide degradation. In another embodiment dephosphorylation of the serine residue of the WW-domain containing polypeptide prevents binding of the WW-domain containing polypeptide and ligand thereby preventing polypeptide degradation. The regulation of protein degradation by the methods of the invention can result in regulation of cell growth. In yet another embodiment modulations in the protein degradation lead to regulation of cell growth. In particular, inhibition of Cdc25 degradation by the ubiquitin pathway results in cell death.
In yet another aspect of the invention relates to a method of treating a WW-domain containing polypeptide-mediated condition in a mammal, wherein the condition results from an alteration in a ligand for the WW-domain containing polypeptide, wherein the ligand is a phosphoserine or phosphothreonine ligand comprising introducing into the mammal an amount of a WW-domain containing polypeptide effective to regulate the ligand, thereby alleviating the condition.
In another embodiment the present invention relates to a method of treating a WW-domain containing polypeptide-mediated condition in a mammal, wherein the condition results from an alteration in the WW-domain containing polypeptide wherein a ligand for the WW-domain contains a phosphoserine or phosphothreonine, comprising introducing into the mammal an amount of a WW-domain containing polypeptide effective to alleviate the condition.
The invention further relates to a method of delivering a drug to treat a condition in a mammal, wherein the condition results from an alteration in a phosphorylated ligand for a WW-domain containing polypeptide, comprising combining the drug and the WW-domain containing polypeptide or a fragment under conditions suitable to form a complex; and administering the complex to the mammal, wherein the complex and phosphorylated ligand interact thereby alleviating the condition.
The inventions which are described herein provide compositions and methods to modulate protein-protein interactions such as binding interactions between signaling or regulatory proteins and their phosphorylated ligands. The methods permit inhibiting or enhancing the interaction between a WW-domain containing polypeptide and its phosphorylated ligand. The methods described herein can be used for regulating cell growth; targeting proteins for cellular degradation; restoring the function of tau to bind microtubules and promote or restore microtubule assembly in neurodegenerative diseases such as Alzheimer""s disease, Dementia pugilistica, Down""s syndrome, Parkinson""s disease, Pick""s disease; identifying a substance which alters the interaction of WW-domain containing polypeptides and their phosphorylated ligands; and targeting drugs to ligands of WW-domain containing polypeptides to treat disease conditions in a mammal. The methods provide a means to assess the interaction of a phosphoserine/phosphothreonine binding module (WW-domain containing polypeptide) and its cellular ligands.